Project Summary/Abstract Penicillin-binding proteins (PBPs) are the enzymes responsible for the biosynthesis of peptidoglycan (PG), the essential glycopolymer component of the bacterial cell wall. PBPs polymerize the monomeric precursor lipid II into PG and covalently crosslink the glycan strands to form a mesh-like network that encases the cell. Despite being known as the protein targets of ?-lactam antibiotics for many years, the precise biochemical functions of PBPs remain poorly understood. The low molecular weight (LMW) PBPs are monofunctional enzymes with uncertain roles in PG synthesis and maturation. LMW PBPs have previously been assumed to act strictly as carboxypeptidases (CPs) that hydrolyze peptide bonds. Our laboratory recently discovered that a LMW PBP from Staphylococcus aureus, PBP4, acts as a robust transpeptidase (TP) instead, catalyzing crosslinking of PG strands both in vitro and in cells. This enzyme is homologous to a number of other LMW PBPs found in Gram-positive pathogens, including species of Enterococci and Streptococci, raising the possibility that some LMW PBPs may be important for PG synthesis in clinically relevant pathogens. A greater fundamental understanding of the functions of LMW PBPs is therefore required. Our laboratory has recently developed methods to extract large amounts of lipid II from bacterial cultures. Access to lipid II makes us uniquely poised to study the TP activity of LMW PBPs. Lipid II can be polymerized into PG strands in vitro enabling us to rapidly evaluate the enzymatic activity of LMW PBPs of interest. Using this lipid II, I have now shown that, like S. aureus PBP4, a LMW PBP from Enterococcus faecalis, PBPX, is also a TP. I observed that PBPX and S. aureus PBP4 share a unique C-terminal beta-strand rich domain (BRD), annotated as DUF1958, that is not present in PBPs that act as CPs. We now propose to explore further the enzymatic functions of LMW PBPs bearing this domain. In Aim 1, we will show that PBPs with the DUF1958 BRD are a previously unrecognized sub-family of LMW enzyme that act as TPs. In Aim 2, we will dissect, in detail, the substrate scope of E. faecalis PBPX and determine if this enzyme has PG crosslinking activity in cells. In Aim 3, we will determine the molecular mechanism by which the DUF1958 BRD directs TP activity. Completion of these aims will define a new class of LMW PBPs that act as TPs, reveal new insights into the role of LMW PBPs in PG synthesis and maturation, and may inform the design of new antibiotics.